Thromb Haemost 2001; 86: 1000-6 © 2001 Schattauer GmbH, Stuttgart
Evidence of α Founder Effect for the Protein C Gene 3363
Inserted C Mutation in Thrombophilic Pedigrees of French
Origin
Patrick Couture
1, Edwin G. Bovill
2, Christine Demers
3, Jacques Simard
4,
Robert Delage
3, Bruce T, Scott
2, Julia E.
Valüere
2, Peter W. Callas
2, Michele Jomphe
5,
Frits R. Rosendaal
6, Martine Aiach
7, George L. Long
21 Department of Infernal Mediane, CHUL Research Center, CHUQ, Quebec, Canada; 2Departments öl Pathology and Biochemistry, University of Vermont, Burlington, USA; 3Department of Hematology, Höpital du Saint-Sacrement, CHA, Quebec, Canada; "Laboratory of Hereditary Cancers, CHUL Research Center,
CHUQ, Quebec, Canada; 5BALZAC Project, University du Quebec, Chicoutimi, Canada; 6Departments of Clinical Epidemiology and Hematology, Leiden University Medical Center, Leiden, The
Netherlands; 7Laboratory of Hemostasis, Broussais Hospital, Paris, France
Keywords Introduction
Protein C deficiency, thrombophilia, genetics Summary
We have previously reported that the 3363 inserted (Ins) C mutation in exon 6 of the protein C gene was present in four unrelated French patients and in four French Canadian families with type I protein C deficiency äs well äs in a large Vermont protein C deficient kindred of French Canadian origin. The present study was designed to investigate the likelihood of the existence of a founder effect for this mutation in protein C deficient individuals of French origin living in France, Quebec and Vermont. In order to demonstrate a possible founder effect for the 3363 InsC mutation, we have previously constructed a htgh-resolution genetic map to locate several highly polymorphic markers dose to the protein C locus. Thereafter, the markers D2S347, D2S2339. D2S383, D2S2271 and D2S2215 were genotyped in 117 heterozygotes from France (n = 7), Quebec (n = 36) or Vermont (n = 74). The allelic frequency distribution of these five markers was also determined in fifty control French Canadian subjects and thirty-two unaffected members of the Vermont kindred with normal protein C levels and compared with their frequency in our cohort of heterozygotes. Our data suggest that patients from Quebec and Vermont carry a common haplotype at the protein C locus. Moreover, in order to study the evolutionary history of the 3363 InsC mutation, we traced back the ascending genealogy of one proband in each of the families with this mutation. These results showed that the 3363 InsC mutation was most probably introduced in Norm America by a couple of French settlers who established themselves in 1669 on Isle d'Orleans located near Quebec City. All heterozygotes for the 3363 InsC mutation living in North America are related to these founders within 10 generations. Thus, these families afford a unique opporturjity to evaluate the role of the protein C System in thrombophilia due to the high degree of linkage disequilibrium at the protein C gene, which in essence holds that variable more constant than in a more heterogeneous population.
Correspondence to: Dr. Patrick Couture, Laval University Medical Center, CHUL Research Center (S-102), 2705 Launer Blvd., Quebec, G1V 4G2, Canada - Tel.: (418) 654-2106; Fax: (418) 654-2277; E-mail: patrick.couture® crchul.ulaval.ca '
Protein C is a vitamin K-dependent glycoprotein and zymogenofa serine-protease which plays an important role in the regulation of thrombin activity. Following proteolytic activation by thrombomodulin-bound thrombin, activated protein C downregulates the coagulation cascade by selective proteolytic inactivation of the procoagulantfactors Va and Villa in the presence of calcium and phospholipids (l, 2). ll also enhances fibrinolysis in part by neutralizing plasminogen acttvator inhibitor-1 by way of the thrombin-activatable fibrinolysis Inhibitor (TAFI). The protein C gene is located on chromosome 2, at positim 2ql3-q21 (3-8), and comprises 9 exons encompassing 11 kb genomfc DNA and encoding a 1795-bp mRNA (9,10).
Protein C deficiency is caused by mutation in the protein C gene and is inherited äs an autosomal codominant trait. More than 190 germliK mutations in the protein C have been described around the world (111 The clinical expression of protein C deficiency is highly variable arid the diagnosis based on plasma measurements of protein C is oft» difficult because of the significant overlap between heterozygotes ad non-carriers. Heterozygous subjects with plasma protein C levefc between 30% and 70% of normal values possess an increased risk oi developing venous thrombosis (12). The prevalence of asymptoniai)' heterozygotes for protein C deficiency in the general population raif» from l in 200 to l in 500 (13,14) while the prevalence of symptomaac heterozygotes ranges from l in 16,000 to l in 32,000 (15, Ife Homozygous or compound heterozygous subjects with plasma proW C levels < 1% are at risk of neonatal purpura fulminans and
venous thrombosis (17). When protein C levels are measuiA | thrombotic manifestations Start later during childhood or early aW
life. ' We have previously reported that the 3363 inserted (Ins) C mulä» i in exon 6 of the protein C gene was present in four unrelated FW* s patients (18) and in four French Canadian families with type IP108*?! deficiency (8) äs well äs in a large Vermont protein C deficient kifl**J of French Canadian origin (19). Thus, the present study was desigtwä* investigate the likelihood of the existence of a founder effect w™j mutation in protein C deficient individuals of French origin ^ί$* France, Quebec and Vermont. Specifically, the demonstratioa^J founder effect for this mutation would provide a great
study the genetic and environmental factors modulating expression of protein C deficiency.
Couture et al.: Protein C Deficiency in Thrombophilic Pedigrees of French Origin
r" — ' ~~
| ghromosome 2 fe|sE83g ,· *'* ¥ 1 {ftromosome bands < / ' LfVntrnmrrc *" Otnelic Distance 134.6 1 Stanford G3 1 Bidlatlon Hybrid Panel 5964'", Human BAC
. fnd Sequences Contig
8AC clones Accessioned sequences RPII-30F3 AC027142.1 RPII-314i.11 AC068282.4 RPII-286H15 AC010976.4
\ •
" m·- rB - HPÜ
"1=Η=ϊ?Ί ^ BigiH s|sH *Θ
I-H :rio^^^^^^^^^^l
1 1 " ' i\\£.\ .1 n, ^ Λ? ^ ^ >£ ^V^ ' A^· ^
o^ "$0^ -vV
Q 1 1 ( 1 135.7 136.8 137.4 138.3 i ' | 6318 1 1 1 t ' ctg 14521 '•·ί^·Β··^···Ι::ιΐ4οο8 Kb
• 188 Kb • 201 Kb • 204 Kbf
PROCf*l / Fine chromosomal localization of Protein C gene. Location in centiMorgan of each markers was determined according to the genetic linkage map of Sseihon and position of the external markers in CentiRay was obtained onto the Stanford G3 radiation hybrid map (resolulion = 41.5 cR 10000 per megabase) ÜB human BAC end sequences contig was obtained by using the Human Genome Project Working Draft of the Washington University Genome Sequencing fiater. St. Louis, MO, USA (http://genome.ucsc.edu/goldenPath/hgTracks.html). The clones come from the Pietcr J. de Jong RPCI-11 librairies (Children's fapilal Oakland - BACPAC Resources Oakland, CA, USA)
Subjects, Materials and Methods
One hundred and seventeen heterozygotes for the 3363 InsC mutation from fnoci in = 7), Quebec (n = 36) or Vermont (n = 74) with type l protein C iffidsncy were analyzed. The diagnosis of type I protein C deficiency was based eAepresenceof plasma levels of protein C antigen below 69% of normal and Iwfevelsof activity by functional assay. The study participants from Vermont
»et drawn from two large related kindreds that consisted of more than 500
«fcmbers over six generations (19) while heterozygotes from Quebec belonged «four large kindreds unrelated to the first or second degree (8). The majority äf Ihese individuals resided in northern Vermont and the Quebec City area. Htierozygotes from France belonged to three small unrelated nuclear families. Maralable family members were contacted and blood sample was taken for |*ftin C measurement and mutation analysis. Fifty control French Canadian rtjects and 32 unaffected members of the Vermont kindreds with normal iBM C levels were also analyzed. All subjects were of French origin. This *ny was approved by the Human Experimentati on Ethics Committee of .' feticipating hospitals and informed consent was obtained from all participants.
'ection
ι Symmetrie PCR was performed äs previously described (8) using the primer
ran'1"· -:· ' "' ~T"~~,GACAACGGCGGCTGCAGG-3' and 6d 5'-Γ*(Ι!<'! ' " · · .· · ( ' ΌΤΑΤΤ-3' for the 3363 InsC mutation. The i. ^* Products were digested with the enzyme Haeül, äs recommended by the
%lier (New England Biolabs, Beverly, MA) and the resulting fragments -•Btsize-separated by electrophoresis on an 1.5% agarose gel.
lite Marker Analyses
Rvemicrosatellite markers close to the protein C locus: D2S347, D2S2339,
Ö"^3,D2S2271 and D2S2215 were genotyped in 117 subjects heterozygous
for the 3363 InsC mutation. The allelic frequency distribution of the.se five markers were also determined in 82 control subjects with normal protein C levels and compared with their frequency in our cohort of heterozygotes. Fig. l gives the fine chromosomal localization of protein C gene. The location in centiMorgan of each markers was determined according to the genetic linkage map of Genethon and position of the external markers in CentiRay was obtained onto the Stanford G3 radiation hybrid map (resolution = 41.5 cR^K,,, per megabase). The human BAC cnd sequences contig was obtained by using the Human Genome Project Working Draft of the Washington University Genome Sequencing Center , St. Louis. MO, USA (http://genome.ucsc.edu/golden-Path/hgTracks.hlml). The clones come from the Pieter J. de Jong RPC1-11 librairies (Children's Hospital Oakland - BACPAC Resources Oakland. CA. USA). The microsatellites were analyzed using PCR and PCR products were size-separated by electrophoresis on an 69r polyacrylamide gcl. The alleles are given äs base pairs.
Genealogical Studie?,
Thromb Haemost 2001; 86: 1000-6
program in histoncal demography database. numerous published marriage reposilories. genealogical dictionaries, etc. The genealogies were analyzed using vanous Software programs developed at the University of Quebec in Chicoutimi (23). The genetic contribution of euch ancestor and the kinship coefficient between probands were calculated äs previously described (24). The genetic contribution of an ancestor represents the sum of transmission probabilities of one gene to each individual of a givcn group and is calculated for each ancestor äs:
ΣΣ(1/2)-ε
' /'
where A is the number of individuals in a given group genealogically related to the ancestor; /; is the number of genealogical paths between the ancestor and the individual; and # is the number of generations separating the ancestor from the individual. for each path. For example. the genetic contribution of a grandfather to his 10 grandchildren is 2.5. The mean genetic contribution per individual is the genetic contribution divided by the number of individuals. On the other hand. the kinship coefficient between two individuals (ß, and B·,)
represents the probability (hat one allele from individual ß, is identical by descent to an allele from individual ß, picked at random at the same locus and is calculated äs:
where A is a common ancestor of ß; and ß,; C is the number of genealogical palhs between ß, and ß:; m(A, C) is the number of generations separating the founder A from the individual ß,, for each path; n(A, C) is the number of generations separating the founder A from the individual ß,, for each path; and
F(A) is the inbreeding coefficient of the founder A. For example, the kinship
coefficient between a grandfather and his grandson is 0.125. The mean kinship coefficient for a group of individuals is the kinship coefficient divided by the total number of pairs of individuals.
Statistical Anahsis
The allele frequencies were compared using the chi-squared lest or Fisher's exact lest where appropriate. These analyses were performed using the JMP statistical Software (release 4.0.1. S AS Institute Ine).
Results
Genetic Basis ofthe Founder Effectfor the 3363 InsC Mutation
In order to investigate the origin of the frameshift mutation 3363 InsC present in Vermont, Quebec and France, we examined allele frequencies of the five microsatellite markers D2S347, D2S2339, D2S383, D2S2271 and D2S2215 which surround and are within 2.6 centiMorgans (cM) of the protein C locus in 117 heterozygotes from Vermont (n = 74), Quebec (n = 36) or France (n = 7). The allelic frequency distribution of these five markers was also determined in fifty control French Canadian subjects and thirty-two unaffected members of the Vermont kindred with normal protein C levels and compared with their frequency in our cohort of heterozygotes (Table l). These results indicated the presence of a common putative haplotype linked to the 3363 InsC mutation in patients from Vermont and Quebec. The common putative haplotype included the alleles 286 (D2S347), 208 (D2S2339), 180 (D2S383), 160 (D2S2271), and 153 (D2S2215). Moreover, segregation analyses in protein C deficient families strongly suggested that 3363 InsC heterozygotes from Vermont and Quebec shared a common mutant allele (Fig. 2). In protein C deficient families from France, however, only the two microsatellite markers (D2S2271, D2S2215) dosest to the protein C locus exhibited identical alleles, most probably because genetic recombinations occurred at the other markers.
Table l Allelic distribution frequencies of microsatellite markers m individuals of French origin living in Quebec and Vermont
D2S347 Allele Quebec N 3363 n=50 n=36 264 00 00 266 00 00 276 .36 .19 278 .04 00 280 00 00 282 00 00 284 .08 00 286 .28 .63' 288 .24 .18 290 00 00 292 00 00 Vermont Allele N 3363 n=32 n=74 .02 00 202 .35 .21 204 00 .01 206 00 00 208 .02 00 210 .02 .01 212 00 .03 .30 .56* .02 .01 .25 .14 .02 .03 D2S2271 Allele Quebec N 3363 n=50 n=36 140 00 . 00 142 .26 .15 144 .02 00 146 .02 .05 148 00 00 150 .04 .03 152 .16 .06 154 .04 .08 156 .28 .03 158 .12 .10 160 .04 .50* 162 .02 00 D2S2339 Quebec N 3363 n=50 n=36 .18 .06 .44 .16 .18 .16 .20 .60* 00 00 00 .02 D2S383 Vermont Allele Quebec N 3363 n=32 n=74 N 3363 n=50 n=36 .14 .06 168 00 00 .31 .18 172 .06 .13 .28 .15 174 .04 00 .27 .60* 176 .20 .11 00 .01 178 .22 .11 00 00 180 .40 .63* 182 .08 .02 184 00 00 Vermont N 3363 n=32 n=74 .20 .18 .14 .02 00 .01 00 00 .02 00 .20 .07 .11 .08 .18 .05 .11 .07 .02 .02 .02 .50* 00 00 D2S22f5 Allele Quebec N 3363 n=50 n=36 139 .04 .06 143 .04 .03 145 .12 .05 147 .12 .10 149 .12 .08 151 .12 .13 153 .16 .47* 155 .12 .05 157 .12 .03 159 .04 00 Vermont N 3363 n=32 n=74 .03 .01 00 .01 .31 .11 .19 .13 .19 .06 .03 .01 .05 ' .52* 00 .01 .14 .09 .06 .05 Vermont N 3363 n=32 n=74 .03 .06 .20 .15 .25 .11 .13 .04 .06 .04 .31 .56* l 00 .03 .02 .01 ( j S j,
Äi p 2« *ga-ga»ng with WM I«»C mutation m m faml'ies tc, Vermont and
tont
Origin of Family's Number of families number 3363 InsC carners
Haplotypes
Microsatellite markers (Cen-»Tel) (D2S .)
Quebec Vermont France Q01 Q02 Q03 Q04 V02 V01 F01 F02 F03 4 1 10 10 5 2 1 56 1 1 1 1 1 1 1 1 1 1 1 2 1 4 2 347 286 286 286 286 286 286 290/284 286 286 286 286 286 286 290/266 292/266 286 286 290/266 290/288 292/266 266/286 268/284 288 2339 208 208 208 208 -208 208 208 208 208 208 208 208 208 206/206 208 208 208 206/204 208 208 202/208 204 204/210 383 180 180 180 180 180 180 180 180 180 180 180 180 180 180 180 178/176 186/172 172/172 176/172 182/172 180/182 170 176 2271 160 160 160 160 160 160 160 160 160 160 160 160 158/140 160 160 160 160 160 160 160 140/160 160 160 2215 153 151/151 153 153 153 153 153 153 157/147 157/149 159/147 149/145 157/147 153 153 153 153 153 153 153 147/153 153 153
6atolog\ oflhe 3363 InsC Mutation m North America
laorder to study the evolutionnary histoiy of the 3363 InsC mutation » North America, we traced back the ascendmg genealogy of one frtand m each of the six families with this mutation We found ten «östors (four married couples and two mdividuals) common to all six fKxetn C deficient families and we hypothesized that each of them «M be at the ongm of the 3363 InsC mutation in the families Then,
m order to deleimme which couple or individual most hkely tepiesented the common ancestor to each proband heterozygous foi the 3363 InsC mutation, we deteimmed the genetic contnbution of those ten ancestois m the six case and four control genealogies (Table 2) The contiol subjects were the spouses of the four Fiench Canadian cases Although all 10 ancestors could be at the ongm of the mutation subjects l and 2 (couple A) have the highest genetic contnbution and theiefore the highest piobabihty of havmg mtroduced the mutation m the Fiench
2 Mean genetic contnbution of the common ancestorb in the six case and fom control genealogies
i i } D s ä |ί | 2 i 3 4 4 5
1
61
8 9 } 10 Ancestors Couple A A B B C C D D Date of mamage 1669 1669 1640 1631 1631 unknown unknown 1627 1628 1628 Place of mamage Quebec Quebec Quebec France France France France France France France Gendei F M F M F M F F F M Cases Mean genetic contnbution per subject (a)(ΧΙΟ"4) 4069 4069 3621 3499 3499 1973 1973 1709 2848 2889 Proportion** 100 100 100 100 100 100 100 100 100 100 Controls* Mean genetic contnbution per subject (b) (ΧΙΟ"4) 000 000 1343 2563 2563 854 854 1465 3662 4089 Proportion** 0 0 75 75 100 75 75 75 100 100 DELTA (a) - (b) (X104) 4069 4069 2279 11 19 11 19 11 19 11 19 244 -814 -1200
jihe control subjects were the spouses of the four first French Canadian cases Proportion of case or control genealogies where the ancestor is found
Thromb Haemost 2001, 86. 1000-6
3363 InsC
1669
J885 l 1895 187! D-rO /O D-r-O D-r-O
1906 D-rO D-rO
α
Proband 4 Proband 2 r, Proband 5 Π-ι-Οα
Proband 6Fig. 3 Genealogy of (he 3363 InsC mutation m North Amenca with year of mär-nage for most couples Pro-bands l and 3 reside m Ver-mont and probands 2. 4,5 and 6 reside in the Quebec City area
Canadian population. Moreover, these two ancestors are present m all 6 genealogies of cases but they are abseilt from those of controls. In fact, ancestors l and 2 have the most specific genetic contribution to cases based on the calculation of the difference m genetic contribution to both groups.
The mean kinship coefficient among the six index cases was also determined at vanous genealogical depths (3, 6, 9 or total number of generations) and compared with that of controls. Table 3 shows that the
mean kinship coefficient is greater among index cases than m controk and mdicates that the probability for index individuals to share an allek identical by descent at the same locus is greater than that of contr* These results showed that the 3363 InsC mutation was most probabl) introduced m North America by a couple of French settlers vd* established themselves m 1669 on Isle d'Orleans located near Quet*·' City. All heterozygotes for the 3363 InsC mutation living in Νοώ
Amenca are related to these founders within ten generations (Fig. 31.
Kinship coefficient (X1Q·4)
Number of generations
Cases 6 Controls* 4 The control subjects wepe the spouses
3 5.2083 0 of the four 6 9 8.6263 12.1494 0 2.346 French Canadian cases.
Total
12.7213 3.0896
Table 3 Meankinship«*
^ A. fcfli'
InsC mutation which was present in four large French .s previously been reported in French patients with type I protein C deficiency and in a large Vermont a high mcidence of venous thrombosis (19, 25). In the tudy fjve highly polymorphic microsatellite markers flanking C locus were used to trace back the origin of the 3363 InsC in Quebec, France and Vermont. The association and """vrfHW anaiyses presented herein indicate that the allelic distribution 4*i me microsatellite markers differs between control subjects and yeejftäous patients for the 3363 InsC mutation suggesting that *seö*"fr°m Quebec, Vermont and possibly from France carry a •αακϋ mutant allele at the protein C locus. The microsatellite marker
igether with the genealogical studies indicate that the 3363 muution must have a single origin, most likely in France. The of recurrent independent mutations is not supported by the of this study. Thus, the 3363 InsC mutation would have been to the French Canadian population äs a result of a founder ifet «hicli is defmed äs "the establishment of a new population by jfe» «inal founders who carry only a small fraction of the total MO*: Variation of the parental population" (26). Some evidence for leafcr effecl in protein C deficiency has already been reported in ätlwpopulations, namely Dutch (27) and Finns (28).
$f\eral demographic factors could have contributed to enhance the f^ilence of the 3363 InsC mutation in the early days of French ·, aament in Canada and during expansion of the new population. The φα of colonization in Norm America coincided with the peak of «antilism in France and in other parts of Europe (29). At that time, fiech emigration to the colomes was not supported and population .fStth was encouraged to fulfill the needs of a trading state for welfare 4is inhabitants. The existence of colonies was perceived äs advanta-gtus to achieve prosperity by providing primary resources and raw Maiais. This policy led to the establishment of measures to restrict •eigration to "La Nouvelle-France" and a series of local incentives , * Ic colony supporting early marriages and high birth rate. The ;«iigration was selective and designed to meet the demands of this ; ffcy. Most of the immigrants were skilled artisans sent for specific sffriods with promise of enhanced Status on their return to France; they ί KR also soldiers encouraged to settle in Canada. The number of immigrants to Canada in the 17lh Century has been estimated to
fWaround 8000 but many of them did not survive long enough to jfsxreate, many returned to France alone or with their families, and •toe were members of religious Orders. Thus, it was estimated that 608 and 1680 which is the period corresponding to the peak •fFrcnch Immigration in "La Nouvelle-France", approximately 3380 ,peeers settled permanently in the St-Lawrence River Valley. This ,aferlimited number of founders constitute the basis for the the French population of today. At that time and following, the birth very high and the growth of the population was rapid (29). •'Ide Champlain founded Quebec City with 30 men in 1608 and, fc firstcensus of the French colony in 1666, there were 3215 people
families. It has been estimated that a little more man 1500 married within the first 50 years of the settlement, contributing a Century later to > 50000 descendants.
"* French settlers and their descendants lived in relative Isolation of conditions imposed by climate, geography, religion, language, «omic Status, political contraints and a rural life-style (30). tended to establish small, stable and self-sufficient communities.
factors favored endogamy. Since the economy was based mostly
on seasonal agriculture and wood industry, population growth quickly exceeded the employment opportunities and between 1840 and 1930, nearly one million French Canadians emigrated to United States, main-ly to Vermont, New Hampshire, Marne and Massachusetts (31). This would explain the presence of the 3363 InsC mutation in the protein C gene and other "French Canadian" mutations responsible for various genetic diseases in New England states (32). Moreover, the early introduction of the 3363 InsC mutation in the Quebec settlement process could imply that this mutation has a high prevalence in the French Canadian population. Epidemiological studies are needed to address this question since the prevalence of protein C deficiency is unknown in the Province of Quebec.
Major advantages may be derived from the knowledge of a founder effect for the 3363 InsC mutation in the New England states and the Province of Quebec, äs is the case for several genetic disorders including familial hypercholesterolemia (33, 34), phenylketonuria (35) and familial hyperchylomicronemia (36) in Quebec. It facilitates screening, genetic counseling, and treatment. It also provides a great opportumty to study (i) gene-gene interactions mvolved in the pathogenesis of thromboembolic disease, (ii) genetic and environmental factors modulating phenotypic expression of protein C gene mutations and (iii) the geographic distribution of this mutation äs well äs population movements.
Addendum
We would like to specify the role each author played m the study. Couture, Bovill, Simard, Long. Aiach, Jomphe and Rosendaal were the researchers mvolved in the design, execution and analysis of the study. Demers and Delage were responsible for the study's conception and design. During the performance of the study, Scott, Valliere and Callas were responsible for daily supervision of the researchers.
Acknowledgementt
This research was supported by grants from the CHUL research center and the Public Health Service Heart, Lung, and Blood Institute, USA, Grant No. PO l HL46703. We are mdebted to the patients and their parenls mvolved in this study. We are also mdebted to Dr. Helene Ve'zina foi genealogical studies.
References
1. Esmon C. Protem-C biochemistry. physiology, and clmical implications. Blood 1983; 62: 1155-8.
2. Marlar R, Kleiss A, Griffin J Mechamsm ot action of human activated protein C, a thrombm-dependent anticoagulant enzyme. Blood 1982; 59. 1067-72.
3. Rocchi M, Roncuzzi L, Santamana R, Archidiacono N, Dente L, Romeo G Mappmg through somatic cell hybnds and cDNA probes of protein C to chromosome 2. factor X to chromosome 13, and alpha 1-acid glycoprotem to chromosome 9. Hum Genet 1986; 74: 30-3.
4. Kalo A, Miura 0. Sumi Y, Aoki N. Assignment of the human protein C gene (PROC) to chromosome region 2ql4-q21 by in situ hybndization. Cytogenet Cell Genet 1988; 47: 46-7.
5. Patracchmi P, Aiello V, Palazzi P, Calzolari E, Bernardi F. Sublocahzation of the human protein C gene on chromosome 2ql3-ql4. Hum Genet 1989; 81: 191-2.
6. Koeleman BP, Reitsma PH, Bakker E, Bertina RM. Location on the human genetic Imkage map of 26 genes mvolved in blood coagulation Thromb Haemost 1997; 77: 873-8.
7 Cox S, Bryant SP, Collms A, Weissenbach J. Doms-Keller H, Koeleman BP, Steinkasserer A, Spurr NK. Integrated genetic map of human chromosome 2. Ann Hum Genet 1995; 59: 413-34
8. Couture P, Demers C, Monssette J, Delage R, Jomphe M, Couture L. Simard J. Type l protein C deficiency m French Canadians: evidence of a
Thromb Haemost 2001; 86: 1000-6
founder effect and association of specific protein C gene mutations with plasma protein C levels. Thromb Haemost 1998; 80: 551-6.
9. Foster DC, Yoshitake S, Davie EW. The nudeotide sequence of the gene for human prolein C. Proc Natl Acad Sei U S A 1985; 82: 4673-7. 10. Plutzky J, Hoskins JA, Long GL, Crabtree GR. Evolution and organization
of the human protein C gene. Proc Natl Acad Sei USA 1986; 83: 546-50. 11. Reitsma PH, Bernardi F, Doig RG. Gandrillc S, Greengard JS, Ireland H,
Krawczak M. Lind B, Long GL. Poort SR, Saito H, Sala N, Witt I, Cooper DN. Protein C deficiency: a database of mutations. 1995 Update. On behalf of the Subcommittee on Plasma Coagulation Inhibitors of the Scientific and Standardizalion Committee of the ISTH. Thromb Haemost 1995; 73: 876-89.
12. Allaart CF. Poort SR. Rosendaal FR. Reitsma PH, Bertina RM, Briet E. Increased nsk of venous thrombosis in carriers of hereditary protein C deficiency defect. Lancet 1993; 341: 134-8.
13. Miletich J, Sherman L, Broze GJ. Absence of thrombosis in subjects with heterozygous protein C deficiency. N Engl J Med 1987; 317: 991-6. 14. Tait RC. Walker ID. Reitsma PH, Islam SI. McCall F. Poort SR, Conkie JA,
Bertina RM. Prevalence of protein C deficiency in the healthy population. Thromb Haemost 1995; 73: 87-93.
15. Gladson CL, Scharrer l, Hach V. Beck KH, Griffin JH. The frequency of type I heterozygous protein S and protein C deficiency in 141 unrelated young patients with venous thrombosis. Thromb Haemost 1988; 59: 18-22. 16. Broekmans AW, Van der Linden IK. Veitkamp JJ, Bertina RM. Prevalence of isolated protein C deficiency in patients with venous thromboembolic disease and in the population. Thromb Haemost 1983; 50: 350a.
17. Marlar RA. Montgomery RR, Broekmans AW. Report on the diagnosis and treatment of homozygous protein C deficiency. Report of the Working Party on Homozygous Protein C Deficiency of the ICTH-Subcommittee on Protein C and Protein S. Thromb Haemost 1989; 61: 529-31.
18. Gandrille S, Aiach M. Identification of mutations in 90 of 121 consecutive symptomatic French patients with a type l protein C deficiency. The French 1NSERM Network on Molecular Abnormalities Responsible for Protein C and Protein S deficiencies. Blood 1995; 86: 2598-605.
19. Tomczak JA, Ando RA, Sobel HG, Bovill EG, Long GL. Genetic analysis of a large kindred exhibiting type l protetn C deficiency and associated thrombosis. Thromb Res 1994; 74: 243-54.
20. Bouchard G. Current issues and new prospects for computerized record linkage in the province of Quebec. Historical Methods 1992; 25: 67-73. 21. Bouchard G, Roy R, Casgrain B, Hubert M. Computer in human sciences:
from family reconstitution to population reconstruction. In: From Information to knowledge: conceptual and content analysis by Computer. Nissan E, Schmidt KM. eds. Intellect, Oxford, UK 1995: 201-7.
22. Jomphe M, Casgrain B. Base de donnees genealogiques RETRO: structure des donnees. Document Balzac IC-181, Chicoutimi (Quebec), Canada, 2000.
23. Jomphe M, Casgrain B, Vezina H. Analyses genealogiques ä partir du fichier RETRO. Document Balzac IC-204, Chicoutimi (Quebec), Canada, 2000.
24. Heyer E. Tremblay M. Variability of the genetic contribution of Quebec population founders associated to some deleterious genes. Am J Hum Genet 1995; 56: 970-8.
25. Bovill EG, Bauer KA, Dickerman JD, Callas P, West B. The clinical spectrum of heterozygous protein C deficiency in a large New England kindred. Blood 1989; 73: 712-7.
26. Mayr E. Animal species and evolution. Cambridge, MA, Harvard University Press, 1963.
27. Reitsma PH, Poort SR, Allaart CF, Briet E, Bertina RM. The spectrum of genetic defects in a panel of 40 Dutch families with symptomatic protein C deficiency type I: heterogeneity and founder effects. Blood 1991; 78: 8904. 28. Levo A, Kuismanen K, Holopainen P, Vahtera E, Rasi V, Krusius T, Partanen J. Single founder mutation (W380G) in type II protein C deficiency in Finland. Thromb Haemost 2000; 84: 424-8.
29. Charbonneau H, Desjardins B, Guillemette A, Landry Y, Legare J, Nault F. Naissance d'une population. Les Frangais etablis au Canada au XVIIe siede. Montreal, Les Presses de l'Universile de Montreal, 1987. 30. Bouchard G, De Braekeleer M. Histoire d'un genöme. Population et
ge'netique dans Test du Quebec. Sillery, Quebec, Presses de l'Universite du Quebec, 1991.
31. Lavoie Y. L'emigration des Quebecois aux Etats-Unis, de 1840 ä 1930. Quebec, Editeur Officiel, 1979.
32. Davignon J, Roy M. Familial hypercholesterolemia in French-Canadians: taking advantage of the presence of a "founder effect". Am J Cardiol 1993; 72:6D-10D.
33. Couture P. Morissette J, Gaudet D, Vohl MC, Gagne C, Bergeron J, Despres JP, Simard J. Fine mapping of low-density lipoprotein receptor gene by genetic linkage on chromosome 19pl3.1-pl3.3 and study of the founder effect of four French Canadian low-density lipoprotein receptor gene mutations. Atherosderosis 1999; 143: 145-51.
34. Gaudet D, Vohl MC, Couture P, Moorjani S, Tremblay G, Perron P, Gagne C, Despres JP. Contribution of receptor negative versus receptor defective mutations in the LDL-receptor gene to angiographically assessed coronary artery disease among young (25-49 years) versus middle-aged (50-64 yearsj men. Atherosderosis 1999; 143: 153-61.
35. John SW, Rozen R, Laframboise R, Laberge C, Scriver CR. Five mutations at the PAH locus account for almost 90% of PKU mutations in French-Canadians from eastern Quebec. Hum Mutat 1992; 1: 72-4.
36. De Braekeleer M, Dionne C, Gagne C, Julien P, Brun D, Ven MM, Lupieo PJ. Founder effect in familial hyperchylomicronemia among French Canadians of Quebec. Hum Hered 1991; 41: 168-73.
Received January 12, 2001 Accepted after revision May 16,2001
Verschijnt wekelijks - Jaargang 19, nr. 6a - 8 februan 2001
vademecu
permanente nascholing huisartsen
Deze uitgave is gerealiseerd met redactionele medewerking van Prof. dr. K. GIN, Alphen a/d Rijn Mevr. A.J. Berendsen, Groningen Prof. dr. M. de Haan, Amsterdam Prof. dr. J.H. Bolk, Leiden Mevr. M.Y.J. van Daelen, Blaricum Prof. dr. Th. B. Voorn, Utrecht
NAECOLOGIE/OBSTETRIE
Wat zijn de huldige
adviezen ten aanzien
van het gebruik van orale
anticonceptiva en het risico
op trombose?
ANTWOORD VAN PROF. DR. F.R. ROSENDAAL, KLINISCH-EPI-DEMIOLOOG, EN DR. F.M. HELMERHORST, GYNAECOLOOG, LEIDS UNIVERSITAIR MEDISCH CENTRUM.
Inleiding
Kort na de introductie van orale anticonceptie werd in 1961 door Jordan in de Lancet melding gemaakt van het optreden van veneuze trombose bij een pilgebruik-ster (1). Hierop volgden meer meldingen en, in de ja-ren zestig en zeventig, ook grote vergelijkende studies waaruit bleek dat het hier inderdaad om een bijwerking van de pil ging. Deze onderzoeken toonden aan dat orale anticonceptiva niet alleen de kans op veneuze trombose verhoogden, maar ook op arteriele aandoe-ningen (2). Recent onderzoek heeft aangetoond dat deze trombotische bijwerkingen van orale anticoncep-tiva nog steeds bestaan (3).
Trombose
Veneuze trombose treedt doorgaans op in de diepe vaten van het been; de trombose ontstaat meestal in een kuitvene en breidt zieh uit tot voorbij de knie of zelfs tot in de lies. Regelmatig (bij circa 20%) treedt embolisering op, waarbij een stolsel via het hart naar de longen wordt vervoerd en aanleiding geeft tot de acute verschijnselen van een longembolie.
Veneuze trombose is een ernstige aandoening: niet alleen is er een letaliteit van 1-2%, maar daarnaast
houdt ongeveer de helft van patienten na een trombo-sebeen last van chronische klachten van het been, het zogenaamde 'posttrombotisch syndroom'.
Bij een kwart van de patienten is dit invaliderend door pijnklachten of ulceratie.
Veneuze trombose
De oorzaken van trombose worden doorgaans verdeeld in verworven en genetische oorzaken (4). Genetische afwijkingen die tot een tromboseneiging leiden (trom-bofilie) zijn ten eerste erfelijke deficienties aan de na-tuurlijke stollingsremmers prote'ine C, prote'ine S of antitrombine. Deze afwijkingen körnen voor bij min-der dan 1% van de bevolking. De tweede groep geneti-sche afwijkingen leiden tot een toegenomen procoagu-lante activiteit. Bij factor V Leiden is stollingsfactor V ongevoelig voor inactivatie door geactiveerd proteme C (APC); men spreekt van APC-resistentie (5). Bij de factor II mutatie (protrombine 2021OA) zijn de plas-maspiegels van factor II verhoogd. Factor V Leiden en factor II mutatie komen voor bij respectievelijk 5% en 2% van de bevolking (4).
Recent zijn overigens nog een aantal protrombotische afwijkingen gevonden die, naar alle waarschijnlijk-heid, van gemengd genetische en verworven oorsprong zijn. Dit zijn hoge niveaus aan stollingsfactor VIII, IX en XI en hyperhomocyste'inemie (4).
Het risico van veneuze trombose is vooral verhoogd bij operaties en immobilisatie. Andere verworven si-tuaties met een verhoogd risico zijn kanker, zwanger-schap, kraambed en het gebruik van vrouwelijke hor-monen, zoals in orale contraceptiva en postmenopau-sale hormoonsuppletie.
Veneuze trombose komt jaarlijks voor bij l op de 1000 mensen. Er is een Sterke leeftijdsgradient, waarbij het risico voor het 40e levensjaar circa l per 10.000 per jaar, en na het 80e jaar ongeveer l per 100 per jaar is. Bij trombofilie, zowel door deficienties van prote'ine C, prote'ine S of antitrombine, als door factor V Leiden of protrombine 2021 OA, is dit risico 5-10 maal hoger (4).
kans op veneuze trotnbose stijgt van l per 10.000 vrouwen per jaar naar 4 per 10.000 vrouwen per jaar. Vanwege de sterke leeftijdstrend van trombose zal het risico wat lager zijn bij de jonge pilgebruikster, en wat hoger bij de oudere pilgebruikster. Enerzijds is dit een in absolute zin bijzonder laag risico, anderzijds is de pil, omdat grote groepen vrouwen deze gebruiken, de belangrijkste oorzaak van veneuze trombose bij jonge vrouwen. Bij vrouwen met erfelijke protrombotische stollingsafwijkingen bestaat een synergistisch effect. Zo is bij pilgebruiksters met factor V Leiden het risico 30 keer hoger (6).
Risico op arteriele trombose
Orale anticonceptiva verhogen eveneens de kans op een arteriele aandoening, zoals een hartinfarct en her-seninfarct (2, 3). Het risico is vooral verhoogd bij vrouwen die ook andere risicofactoren voor arteriele aandoeningen hebben, waarvan de belangrijkste zijn roken, diabetes mellitus, hypertensie en hypercholeste-rolemie. Vooral voor roken is een synergistisch effect aangetoond. Deze factoren zijn risicofactoren voor atherosclerose, en speien daarom geen rol in de etiolo-gie van veneuze trombose. De protrombotische stol-hngsfactorafwijkingen die het risico van veneuze trombose zo sterk verhogen, hebben slechts een gering effect op het optreden van arteriele aandoeningen. Hoewel het hartinfarct een grotere letal iteit heeft dan veneuze trombose (10% versus 2%), is de incidentie bij jonge vrouwen aanzienlijk lager dan van veneuze trombose, zo zeer zelfs dat veneuze trombose tot meer sterfgevallen bij jonge vrouwen leidt dan arteriele trombose. Zo rond het 30-35e jaar draait dit om.
Omgaan met kleine risico's
Bij het omgaan met risico's speien twee elementen een belangrijke rol: ten eerste informatieverschaffing en ten tweede de afweging tegen mogelijke nadelen en tegen de voor- en nadelen van alternatieven. Bij de afweging over wel versus geen pilgebruik wordt de effectiviteit en het gemak afgezet tegen de kans op bijwerkingen. Dit is enigszins een afweging tussen onvergelijkbare grootheden en daarom individueel en subjectief. Hier is een goede informatie aan de ge-bruikster het belangrijkst. De afgelopen decennia heb-ben veel vrouwen deze afweging door laten slaan ten faveure van pilgebruik. Veel eenvoudiger is de keuze tussen verschallende types monofasische orale anticon-ceptiva: aange-zien er geen verschillen in effectiviteit zijn, dient het
preparaat met de laagste frequentie van bijwerkingen te worden gekozen.
Soorten orale anticonceptiva
De monofasische combinatiepreparaten worden verre-weg het meest gebruikt. We zullen ons hier beperken tot deze combinatiepreparaten. Er zijn geen goed gedo-cumenteerde voordelen wat betreft werking en bijwer-kingen voor de andere combinatiepiltypen, zoals bifa-sische en trifabifa-sische preparaten en middelen die uit-sluitend progestageen bevatten ('progestin-only'). De eerste anticonceptiva die op de markt kwamen be-gin jaren 60 bevatten een hoge dosis oestrogeen, 100 tot 150 microgram (meestal ethinyloestradiol) en een eerste generatie progestageen (zoals lynestrol). In het streven naar veiliger anticonceptiva zijn er twee ont-wikkelingen geweest. Ten eerste is de dosis oestrogeen verlaagd, eerst naar 50 microgram, toen 30 microgram en thans zijn er anticonceptiva op de markt met 20 of minder microgram oestrogeen. Aan de oestrogene component zelf is niet veel veranderd, dit is ethinyl-oestradiol gebleven. Wat betreft de progestagene com-ponent zijn er marginale veranderingen in de dosis geweest. De belangrijkste verandering was de intro-ductie van nieuwe progestagenen. Deze worden aange-duid met 'generaties'. In de jaren 70 kwam de tweede generatie progestagenen, waartoe levonorgestrel be-hoort. Sinds midden jaren tachtig zijn er orale anticon-ceptiva met een derde generatie progestageen, dit zijn de Stoffen desogestrel en gestodeen. Op het ogenblik zijn de meest voorgeschreven orale anticonceptiva die met 30 microgram ethinyloestradiol, met een ongeveer gelijke verdeling over tweede- en derde generatie pro-gestagenen.
ge-doseerde anticonceptiva, lijkt er weinig reden te zijn om orale anticonceptiva met minder dan 30 microgram ethinyloestradiol voor te schrijven.
Effect van verandering van de
progestage-ne compoprogestage-nent
In 1995 bleek in een aantal studies, waaronder een Ne-derlandse, dat orale anticonceptiva met een derde ge-neratie progestageen het risico van veneuze trombose verhoogden en zelfs tweemaal vaker trombose veroor-zaakten dan orale anticonceptiva met een tweede gene-ratie progestageen (7, 8). Het gaat hierbij om anticon-ceptiva die desogestrel of gestodeen bevatten. Recent bleek in een prospectieve farmaco-epidemiologische Studie uit Utrecht dat het risico vooral was verhoogd, tot zeven maal, bij de jongste gebruikers (9). Deze risi-co's zijn gesuperponeerd op het viervoudig verhoogde risico van tweede generatie contraceptiva. Omdat deze bevindingen onverwacht waren, gaven zij aanleiding tot enige controverse. Onderzoek uit Maastricht hielp de verhoogde risico's te verklaren, aangezien bleek dat anticonceptiva met een derde generatie progestageen de bloedstolling ernstiger verstoorden dan anticoncep-tiva met een tweede generatie progestageen (10). De derde generatie progestagenen waren echter ont-wikkeld om het risico van arteriele ziekten te vermin-deren. Inderdaad bleken zij„bij gezonde gebruiksters tot een verbetering van het lipidenprofiel te leiden, met een verhoging van het HDL-cholesterol. De vraag is of dit zieh vertaalt in een verminderde kans op een hartin-farct. Een groot Engels onderzoek toonde aan dat er geen gunstig effect was op het optreden van een hart-infarct(ll).
Anticonceptie bij vrouwen met
risicofacto-ren
Bij vrouwen met risicofactoren voor zowel arteriele als veneuze trombose draagt het gebruik van orale anti-conceptie bij aan verdere verhoging van het risico. Voor veneuze trombose betreft dit ten eerste vrouwen met een voorgeschiedenis van veneuze trombose, en ten tweede vrouwen met een mogelijke erfelijke trom-boseneiging, dat wil zeggen met factor V Leiden of een positieve familiegeschiedenis voor trombose. Het risico van arteriele trombose stijgt vanaf het 30-35e jaar en daarom is het van belang de combinatie pilge-bruik en roken te vermijden bij vrouwen boven deze leeftijd. Ongecontroleerde hypertensie, hypercholeste-rolemie en diabetes zijn contra-indicaties voor orale anticonceptie. Geen van deze contra-indicaties is abso-luut omdat er soms geen adequate alternatieven zijn. De belangrijkste contra-indicatie is een eerdere trom-bose, aangezien de recidiefkans na een eerste trombose enige procenten per jaar is. Voor vrouwen met risico-factoren zullen sociale omstandigheden mede bepalend zijn of toch voor een oraal anticonceptivum wordt ge-kozen. Hierbij moet men ook naar de leeftijd van de vrouw kijken: bij jonge vrouwen is de kans op trombo-se in absolute termen zeer klein, en blijft klein zelfs wanneer verscheidene risicofactoren aanwezig zijn. Nederland is overigens een van de weinige landen waar grote aantallen vrouwen boven de 35 jaar orale anticonceptiva gebruiken.
Samenvatting
microgram ethinyloestradiol. Preparaten met een derde generatie progestageen (desogestrel, gestodeen) verbo-gen de kans op veneuze trombose, zonder een vermin-derde kans op arteriele trombose, en dienen daarom te worden vermeden, zeker bij vrouwen die met de pii beginnen. Het eerder doorgemaakt hebben van een veneuze of arteriele trombose is een Sterke con-tra-indicatie voor orale anticonceptie. Aanwezigheid van risicofactoren voor trombose, vooral een erfelijke tromboseneiging (factor V Leiden, deficienties van proteine C, prote'fne S of antitrombine) vormen een contra-indicatie voor orale anticonceptie.
Referenties:
1 Jordan WM. Pulmonary embolism. Lancet 1961; ii: 1146-47.
2 Stadel BV. Oral contraceptives and cardiovascu-lar disease (first of two parts).
N EnglJMed 1981; 305: 612-18.
3 Cardiovascular disease and steroid hormone con-traception. Report of a WHO Scientific group. WHO Technical Report Series, no. 877. World Health Organization. Geneva 1998.
4 Rosendaal FR. Venous thrombosis: a multicausal disease. Lancet 1999; 353: 1167-73.
5 Rosendaal FR. Onlangs ontdekte frequente oor-zaak van veneuze trombose: factor V Leiden, een gemuteerde factor V, resistent legen inactivering door prote'fne C.
Ned Tijdschr Geneeskd 1994; 138: 1944-48.
6 Vandenbroucke JP, Koster T, Briet E, Reitsma PH, Bertina RM, Rosendaal FR. Increased risk of venous thrombosis in oral-contraceptive users who are carriers of factor V Leiden mutation. Lancet 1994; 344: 1453-57.
7 Bloemenkamp KWM, Rosendaal FR, Heimer-horst FM, Büller HR, Vandenbroucke JP.
Enhancement by factor V Leiden mutation of risk of deep-vein thrombosis associated with oral con-traceptives containing a third-generation progesta-gen. Lancet 1995; 346: 1593-96.
8 World Health Organization. Effect of different progestagens in low oestrogen oral contraceptives on venous thromboembolic disease. World Health Organization Collaborative Study of Cardiovascu-lar Disease and Steroid Hormone Contraception. Lancet 1995; 346: 1582-88.
9 Herings RMC, Urquhart J, Leufkens HGM. Venous thromboembolism among new users of different oral contraceptives.
Lancet 1999; 354: 127-28.
10 Rosing J, Tans G, Nicolaes GA, et al.
Oral contraceptives and venous thrombosis: diffe-rent sensitivities to activated protein C in women using second- and third-generation oral contra-ceptives. Br J Haematol 1997; 97: 233-38. 11 Dünn N, Thorogood M, Faragher B, et al. Oral
contraceptives and myocardial infarction: results of the MICA case-control study.
